TY - JOUR
T1 - Expression and characterization of elastase inhibitors from the ascarid nematodes Anisakis simplex and Ascaris suum
AU - Nguyen, Tien T.
AU - Qasim, M. A.
AU - Morris, Stephen
AU - Lu, Cheng Chan
AU - Hill, Dolores
AU - Laskowski, Michael
AU - Sakanari, Judy A.
N1 - Funding Information:
We thank Paul Johnson and Tom Worthington, Monterey Fish, San Francisco for providing us with specimens, Dick Winant and Dr A. Smith, Protein and Nucleic Acid Facility, Stanford, CA for their excellent work in protein sequencing, and the Department of Pathology, UCSF for its support. This work was funded by NIH GM10831 grant to ML at Purdue University and an NIH First Award AI29457 and an Affirmative Action Faculty Development Award from the Academic Senate, University of California, San Francisco to JAS.
PY - 1999/7/30
Y1 - 1999/7/30
N2 - Two elastase inhibitors, ASPI-1 and ASPI-2, from the parasitic nematode Anisakis simplex, have been isolated and characterized. Because these inhibitors are similar in size (60 amino acids in length) and primary sequence (52 and 47% identical) to the Ascaris suum chymotrypsin/elastase inhibitor-1 (AsC/E-1), we suggest that these Anisakis elastase inhibitors belong to the same unique class of canonical inhibitors formed by the family of Ascaris inhibitors (Huang K, Strynadka NCJ, Bernard VD, Peanasky RJ, James MG. Structure 1994;2:679-689). To compare ASPI-1 with AsC/E-1, we expressed both inhibitors in Pichia pastoris and found that: (1) the association constant of rASPI-1 with porcine pancreatic elastase (PPE) is similar to native inhibitor (K(a)=4.5x109 and 6.4x109 M-1, respectively); (2) rASPI-1 is a potent inhibitor of PPE and human leukocyte elastase (K(a)=1.6x109 M-1); and (3) it is only a very weak inhibitor of chymotrypsin (CHYM) (K(a)=1.2x106 M-1). In contrast to the Anisakis inhibitor, however, rAsC/E inhibitor-1 is a very strong inhibitor of both PPE (K(a)=3.5x1010 M-1) and CHYM (K(a)=3.6x1012 M-1). We also found that the determined reactive sites (P1-P1') of rASPI-1 and rAsC/E-1, as recognized by PPE, are Ala 28-Met 29 and Leu 31-Met 32, respectively. These P1-P1' residues of AsC/E-1 constitute the same reactive site as that also recognized by CHYM (Peanasky RJ, Bentz Y, Homandberg GA, Minor ST, Babin DR. Arch Biochem Biophys 1994;232:135-142). The difference in specificities of ASPI-1 and AsC/E-1 toward their cognate serine proteases may be attributed to the P1 and P3' residues in the inhibitors. Elastase, which recognizes both alanine and leucine, can accommodate both ascarid inhibitors, whereas chymotrypsin, which prefers bulky, hydrophobic residues, only recognizes the Ascaris C/E inhibitor-1. Copyright (C) 1999 Elsevier Science B.V.
AB - Two elastase inhibitors, ASPI-1 and ASPI-2, from the parasitic nematode Anisakis simplex, have been isolated and characterized. Because these inhibitors are similar in size (60 amino acids in length) and primary sequence (52 and 47% identical) to the Ascaris suum chymotrypsin/elastase inhibitor-1 (AsC/E-1), we suggest that these Anisakis elastase inhibitors belong to the same unique class of canonical inhibitors formed by the family of Ascaris inhibitors (Huang K, Strynadka NCJ, Bernard VD, Peanasky RJ, James MG. Structure 1994;2:679-689). To compare ASPI-1 with AsC/E-1, we expressed both inhibitors in Pichia pastoris and found that: (1) the association constant of rASPI-1 with porcine pancreatic elastase (PPE) is similar to native inhibitor (K(a)=4.5x109 and 6.4x109 M-1, respectively); (2) rASPI-1 is a potent inhibitor of PPE and human leukocyte elastase (K(a)=1.6x109 M-1); and (3) it is only a very weak inhibitor of chymotrypsin (CHYM) (K(a)=1.2x106 M-1). In contrast to the Anisakis inhibitor, however, rAsC/E inhibitor-1 is a very strong inhibitor of both PPE (K(a)=3.5x1010 M-1) and CHYM (K(a)=3.6x1012 M-1). We also found that the determined reactive sites (P1-P1') of rASPI-1 and rAsC/E-1, as recognized by PPE, are Ala 28-Met 29 and Leu 31-Met 32, respectively. These P1-P1' residues of AsC/E-1 constitute the same reactive site as that also recognized by CHYM (Peanasky RJ, Bentz Y, Homandberg GA, Minor ST, Babin DR. Arch Biochem Biophys 1994;232:135-142). The difference in specificities of ASPI-1 and AsC/E-1 toward their cognate serine proteases may be attributed to the P1 and P3' residues in the inhibitors. Elastase, which recognizes both alanine and leucine, can accommodate both ascarid inhibitors, whereas chymotrypsin, which prefers bulky, hydrophobic residues, only recognizes the Ascaris C/E inhibitor-1. Copyright (C) 1999 Elsevier Science B.V.
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U2 - 10.1016/S0166-6851(99)00088-2
DO - 10.1016/S0166-6851(99)00088-2
M3 - Article
C2 - 10477178
AN - SCOPUS:0032808666
VL - 102
SP - 79
EP - 89
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
SN - 0166-6851
IS - 1
ER -